IntroductionHuangma Tincture (HMT) is a Chinese patent medicine with a history of clinical use for more than 60 years, widely used for treating dermal chronic ulcer such as diabetic foot ulcer. However, the overall quality evaluation and control method of HMT has not yet been researched.ObjectiveThe aim of this study is to establish a comprehensive quality evaluation and control method for HMT based on high‐performance liquid chromatography (HPLC) fingerprint, chemometrics, and multicomponent content determination.MethodsEstablishing chemical fingerprint of HMT and carrying out similarity analysis comprehensively reflect the consistency of the formulation in terms of chemical composition. Chemometrics analyses, including hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares‐discriminant analysis (OPLS‐DA), were performed to identify components crucial to quality differences. The content of five potential bioactive ingredients (brucine, strychnine, jatrorrhizine, coptisine, and berberine hydrochloride) was determined as a supplementary quality control measure.ResultsHPLC fingerprint of HMT with similarity index > 0.990 was established, in which five common compounds (brucine, strychnine, jatrorrhizine, coptisine, and berberine hydrochloride) were identified. HCA, PCA, and OPLS‐DA results, validated through 200 permutation tests, were basically consistent. The contents of brucine, strychnine, jatrorrhizine, coptisine, and berberine hydrochloride in 20 batches of HMT samples were 0.2088–0.5556, 0.2599–0.9868, 0.1358–0.2092, 0.2634–0.6843, and 1.8301–2.7826 mg/mL, respectively.ConclusionHPLC fingerprint combined with chemometrics and multicomponent content determination considering both effect and toxicity provides a robust method for the comprehensive quality evaluation and control of HMT.